Acta Pharmacologica Sinica最新文献

Patients suffering epilepsy caused by the gain-of-function mutants of the hKCNT1 potassium channels are drug refractory. In this study, we cloned a novel human KCNT1B channel isoform using the brain cDNA library and conducted patch-clamp and molecular docking analyses to characterize the pharmacological properties of the hKCNT1B channel using thirteen drugs. Among cinchona alkaloids, we found that hydroquinine exerted the strongest blocking effect on the hKCNT1B channel, especially the F313L mutant. In addition, we confirmed the antitussive drug tipepidine was also a potent inhibitor of the hKCNT1B channel. Subsequently, we proved that these two drugs produced an excellent therapeutic effect on the epileptic model of KCNT1 Y777H mutant male mice; thus, both could be ready-to-use anti-epileptic drugs. On the other hand, we demonstrated that the activation of the KCNT1 channel by loxapine and clozapine was through interacting with pore domain residues to reverse the run-down of the KCNT1 channel. Taken together, our results provide new insights into the mechanism of the modulators in regulating the KCNT1 channel activity as well as important candidates for clinical tests in the treatment of KCNT1 mutant-associated epilepsy.

Diabetic cardiomyopathy causes end-stage heart failure, resulting in high morbidity and mortality in type 2 diabetes mellitus (T2DM) patients. Long-term treatment targeting metabolism is an emerging field in the treatment of diabetic cardiomyopathy. Semaglutide, an agonist of the glucagon-like peptide 1 receptor, is clinically approved for the treatment of T2DM and provides cardiac benefits in patients. However, the cardioprotective mechanism of semaglutide, especially its direct effects on cardiomyocytes (CMs), is not fully understood. Here, we used 8-week diabetic and obese db/db mice treated with semaglutide (200 μg·kg·d^-1, i.p.) to study its direct effect on CMs and the underlying mechanisms. Our results revealed that the consecutive application of semaglutide improved cardiac function. Increased AMPK and ULK1 phosphorylation levels were detected, accompanied by elevated [Ca²⁺]_mito. Seahorse analysis revealed that semaglutide increases ATP production via elevated basal and maximum respiration rates as well as spare respiration capacity in CMs. Transmission electron microscopy revealed improved mitochondrial morphology in the cardiomyocytes of db/db mice. On the other hand, Western blot analysis revealed increased Parkin and LC3 protein expression, indicating mitophagy in CMs. Collectively, our findings demonstrate that semaglutide directly protects CMs from high-glucose damage by promoting AMPK-dependent ATP production as well as ULK1-mediated mitophagy in db/db mice.

Eukaryotic translation initiation factor 4A1 (eIF4A1) is an ATP-dependent RNA helicase that participates in a variety of biological and pathological processes such as cell proliferation and apoptosis, and cancer. In this study we investigated the role of eIF4A1 in ischemic heart disease. The myocardial ischemia/reperfusion (I/R) model was established in mice by ligation of the left anterior descending artery for 45 min with the subsequent reperfusion for 24 h; cultured neonatal mouse ventricular cardiomyocytes (NMVCs) treated with H₂O₂ (200 μM) or H/R (12 h hypoxia and 12 h reoxygenation) were used for in vitro study. We showed that the expression levels of eIF4A1 were significantly increased in I/R-treated myocardium and in H₂O₂- or H/R-treated NMVCs. In NMVCs, eIF4A1 overexpression drastically enhanced LDH level, caspase 3 activity, and cell apoptosis. eIF4A1 overexpression also significantly reduced anti-apoptotic protein Bcl2 and elevated pro-apoptotic protein Bax expression, whereas eIF4A1 deficiency produced the opposite responses. Importantly, cardiomyocyte-specific eIF4A1 knockout attenuated cardiomyocyte apoptosis, reduced infarct area, and improved cardiac function in myocardial I/R mice. We demonstrated that eIF4A1 directly bound to transgelin (Tagln) to prevent its ubiquitination degradation and subsequent up-regulation of p53, and then promoted nuclear translocation of Tagln and p53. Nuclear localization of Tagln and p53 was increased in H₂O₂-treated NMVCs. Silencing Tagln reversed the pro-apoptotic effects of eIF4A1. Noticeably, eIF4A1 exerted the similar effects in AC16 human cardiomyocytes. In conclusion, eIF4A1 is a detrimental factor in myocardial I/R injury via promoting expression and nuclear translocation of Tagln and p53 and might be a potential target for myocardial I/R injury. This study highlights a novel biological role of eIF4A1 by interacting with non-translational-related factor Tagln in myocardial I/R injury.

Dysregulation of long non-coding RNAs (lncRNAs) is common in colorectal cancer liver metastasis (CRLM). Emerging evidence links lncRNAs to multiple stages of metastasis from initial migration to colonization of distant organs. In this study we investigated the role of lncRNAs in metabolic reprogramming during CRLM using patient-derived organoid (PDO) models. We established five pairs of PDOs from primary tumors and matched liver metastatic lesions, followed by microarray analysis. We found that USP3-AS1 was significantly upregulated in CRLM-derived PDOs compared to primary tumors. High level of USP3-AS1 was positively associated with postoperative liver metastasis and negatively correlated with the prognosis of colorectal cancer (CRC) patients. Overexpression of USP3-AS1 significantly enhanced both sphere formation efficiency and liver metastasis in PDOs. Gene set enrichment analysis revealed that USP3-AS1 upregulation significantly enriched glycolysis and MYC signaling pathways. Metabolomics analysis confirmed that USP3-AS1 promoted glycolysis in PDOs, whereas glycolysis inhibition partially attenuated the effects of USP3-AS1 overexpression on PDO growth and liver metastasis. We revealed that USP3-AS1 stabilized MYC via post-translational deubiquitination, thereby promoting glycolysis. We demonstrated that USP3-AS1 increased the stability of USP3 mRNA, resulting in higher USP3 protein expression. The elevated USP3 protein then interacted with MYC and promoted its stability by deubiquitination. The USP3-AS1-MYC-glycolysis regulatory axis modulated liver metastasis by promoting H3K18 lactylation and CDC27 expression in CRC. In conclusion, USP3-AS1 is a novel promoter of CRLM by inducing histone lactylation.

Cancer heterogeneity, characterized by diverse populations of tumorigenic cells, involves the occurrence of differential phenotypes with variable expressions of receptor tyrosine kinases. Aberrant expressions of mesenchymal-epithelial transition (MET) and recepteur d'origine nantais (RON) receptors contribute to the phenotypic heterogeneity of cancer cells, which poses a major therapeutic challenge. This study aims to develop a dual-targeting antibody-drug conjugate (ADC) that can act against both MET and RON for treating cancers with high phenotypic heterogeneity. Through immunohistochemical staining, we show that MET and RON expressions are highly heterogeneous with differential combinations in more than 40% of pancreatic and triple-negative breast cancer cases. This expressional heterogeneity provides the rationale to target both receptors for cancer therapy. A humanized bispecific monoclonal antibody specific to both MET and RON (PCMbs-MR) is generated through IgG recombination using monoclonal antibody sequences specific to MET and RON, respectively. Monomethyl auristatin E is conjugated to PCMbs-MR to generate a dual-targeting ADC (PCMdt-MMAE), with a drug-to-antibody ratio of 4:1. Various cancer cell lines were used to determine PCMdt-MMAE-mediated biological activities. The efficacy of PCMdt-MMAE in vivo is evaluated using multiple xenograft tumor models. PCMdt-MMAE shows a favorable pharmacokinetic profile, with a maximum tolerated dose of ~30 mg/kg in mice. Toxicological studies using Sprague-Dawley rats reveal that PCMdt-MMAE is relatively safe with slight-to-moderate, temporary, and reversible adverse events. Functionally, PCMdt-MMAE induces a robust internalization of both MET and RON and causes a large-scale cell death in cancer cell lines exhibiting MET and RON heterogeneous co-expressions. Both in vitro and in vivo studies demonstrate that the dual-targeting approach in the form of an ADC is highly effective with a long-lasting effect against tumors exhibiting MET/RON heterogeneous phenotypes. Hence, we can suggest that a dual-targeting ADC specific to both MET and RON can be employed as a novel therapeutic strategy for tumors with expressional phenotypic heterogeneity.

Hypoxia is a key feature of the tumor microenvironment that leads to the failure of many chemotherapies and induces more aggressive and resistant cancer phenotypes. Up to date, there are very few compounds and treatments that can target hypoxia. BE-43547A₂ from Streptomyces sp. was one of the most hypoxia-selective compounds against PANC-1, MCF-7, and K562 cell lines. In this study, we investigated the molecular mechanism underlying the hypoxia selectivity of BE-43547A₂ in human pancreatic cancer cells. We showed that BE-43547A₂ displayed hypoxia-selective cytotoxicity in five pancreatic cancer cells (PANC-1, Capan-2, MIA PaCa-2, AsPC-1, and PaTu8988T) with IC₅₀ values under hypoxia considerably lower than those under normoxia. We demonstrated that BE-43547A₂ is directly bound to eEF1A1 protein in PaTu8988T cells under hypoxia. Furthermore, we revealed that hypoxia significantly elevated the expression levels of HIF1α, FoxO1, and eEF1A1 in the five pancreatic cancer cells; eEF1A1 interacted with FoxO1 in the cytoplasm, which was disrupted by BE-43547A₂ followed by the nuclear translocation of FoxO1 and ultimate inhibition of JAK/STAT3 signaling pathway under hypoxia. This study reveals that BE-43547A₂, targeting eEF1A1, disrupts its interaction with FoxO1 in human pancreatic cancer cells under hypoxia. This compound could serve as a potential hypoxia-selective therapy.

Vascular smooth muscle cell (VSMC) phenotypic switching plays a crucial role in the initiation and progression of atherosclerosis. Dehydrocorydaline (DHC), a major active component of the traditional Chinese herbal medicine Rhizoma Corydalis, exhibits diverse pharmacological effects. However, its impact on VSMCs remains largely unknown. This study aims to investigate the effects and underlying mechanisms of DHC in phenotypic switching of VSMCs. Our study revealed that DHC increased the mRNA and protein levels of rat VSMC contractile phenotype markers, such as calponin 1 (Cnn1), myosin heavy chain (Myh11, SM-MHC), smooth muscle 22α (Sm22α), and alpha-smooth muscle actin (Acta2, α-SMA) in a time- and dose-dependent manner. Additionally, DHC inhibited platelet-derived growth factor-BB-induced VSMC proliferation and migration. In Apoe^-/- mice, DHC treatment resulted in reduced carotid plaque areas and macrophage infiltration, along with increased contractile phenotype marker expression. RNA sequencing analysis revealed a significant upregulation of spectrin alpha, erythrocytic 1 (Spta1) in DHC-treated rat VSMCs. Strikingly, Spta1 knockdown effectively negated the increase in contractile phenotype marker expression in VSMCs that was initially prompted by DHC. Therefore, DHC preserves the VSMC contractile phenotype through Spta1, thereby attenuating carotid artery atherosclerotic plaques in Apoe^-/- mice. This study provides evidence supporting the potential use of Chinese herbal medicines, particularly those containing DHC such as Rhizoma Corydalis, in the treatment of atherosclerotic cardiovascular disease, thus expanding the clinical application of such herbal remedies.

Increased level of angiotensin II (Ang II) plays a central role in the development of hypertensive vascular remodeling. In this study, we identified the deubiquitinating enzyme Josephin domain-containing protein 2 (JOSD2) as a protective factor and investigated its molecular mechanism in Ang II-induced vascular remodeling. First, we found that JOSD2 was upregulated in aortic smooth muscle cells, but not in endothelial cells of Ang II-challenged mouse vascular tissues. Whole-body knockout of JOSD2 significantly deteriorated Ang II-induced vascular remodeling in mice. Conversely, Ang II-induced vascular remodeling was reversed by vascular smooth muscle cell (VSMC)-specific JOSD2 overexpression. In vitro, JOSD2 deficiency aggravated Ang II-induced fibrosis, proliferation, and migration VSMCs, while these changes were reversed by JOSD2 overexpression. RNA-seq analysis showed that the protective effects of JOSD2 in VSMCs were related to the TGFβ-SMAD pathway. Furthermore, the LC-MS/MS analysis identified SMAD7, a negative regulator in the TGFβ-SMAD pathway, as the substrate of JOSD2. JOSD2 specifically bound to the MH1 domain of SMAD7 to remove the K48-linked ubiquitin chains from SMAD7 at lysine 220 to sustain SMAD7 stability. Taken together, our finding reveals that the JOSD2-SMAD7 axis is critical for relieving Ang II-induced vascular remodeling and JOSD2 may be a novel and potential therapeutic target for hypertensive vascular remodeling.

The bromodomain (BRD) represents a highly conserved structural module that provides BRD proteins with fundamental functionality in modulating protein-protein interactions involved in diverse biological processes such as chromatin-mediated gene transcription, DNA recombination, replication and repair. Consequently, dysregulation of BRD proteins has been implicated in the pathogenesis of numerous human diseases. In recent years, considerable scientific endeavors have focused on unraveling the molecular mechanisms underlying BRDs and developing inhibitors that target these domains. While these inhibitors compete for binding with the acetylated lysine binding site of BRDs, achieving inhibition of BRD proteins via competitive pocket binding has proven challenging due to the conserved nature of these pockets. To address this limitation, the present study employed dynamic simulations for a comprehensive analysis, leading to the identification of a non-conserved pocket in CECR2 for achieving BRD family inhibition through allosteric modulation. Subsequently, the compound BAY 11-7085 was proven capable of covalently binding to C494 of this pocket after covalent docking and biological verification in vitro. The allosteric inhibition strategy of CECR2 was further verified by the structurally optimized compound LC-CE-7, which is an allosteric covalent CECR2 inhibitor with anti-cancer effects in MDA-MB-231 cells.

The hepatoprotective effect of the fruit of Lycium barbarum has been documented in China over millennia. Lycium barbarum polysaccharides (LBPs) were the first macromolecules reported to mitigate liver fibrosis in carbon tetrachloride (CCl₄)-treated mice. Herein, a neutral peptidoglycan, named as LBPW, was extracted from the fruit of Lycium barbarum. In this study, we investigated the hepatoprotective mechanisms of LBPW. CCl₄-induced liver fibrosis mice were administered LBPW (50, 100, 200 mg ·kg^-1 ·d^-1, i.p.) or (100, 200, 300 mg· kg^-1 ·d^-1, i.g.) for 6 weeks. We showed that either i.p. or i.g. administration of LBPW dose-dependently attenuated liver damage and fibrosis in CCl₄-treated mice. Pharmacokinetic analysis showed that cyanine 5.5 amine (Cy5.5)-labeled LBPW (Cy5.5-LBPW) could be detected in the liver through i.p. and i.g. administration with i.g.-administered Cy5.5-LBPW mainly accumulating in the intestine. In TGF-β1-stimulated LX-2 cells as well as in the liver of CCl₄-treated mice, we demonstrated that LBPW significantly upregulated Smad7, a negative regulator of TGF-β/Smad signaling, to retard the activation of hepatic stellate cells (HSCs) and prevent liver fibrosis. On the other hand, LBPW significantly boosted the abundance of Akkermansia muciniphila (A. muciniphila) and fortified gut barrier function. We demonstrated that A. muciniphila might be responsible for the efficacy of LBPW since decreasing the abundance of this bacterium by antibiotics (Abs) blocked the effectiveness of LBPW. Overall, our results show that LBPW may exert the hepatoprotective effect via rebalancing TGF-β/Smad7 signaling and propagating gut commensal A. muciniphila, suggesting that LBPW could be leading components to be developed as new drug candidates or nutraceuticals against liver fibrosis.